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In this study, a mixed microalgal culture grown in secondarily treated domestic wastewater effluent was investigated for biodiesel production using in situ transesterification method with conventional heating. The total lipid content of the mixed culture was found as 26.2% ± 0.6 by weight of dry biomass, and 74% of the lipids were contributed by total glycerides. In situ transesterification with conventional heating process under acidic conditions produced higher biodiesel yield with chloroform as the co-solvent (82.1% ± 3.9) compared to hexane (55.3% ± 3.9) under the same reaction conditions. The gas chromatography analysis showed that FAME composition was mainly composed of palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acid methyl esters., and thus the mixed microalgal culture fed by domestic wastewaters has had comparable biodiesel conversion yields and FAME composition to mono-culture and pure cultures fed by synthetic culture media. Hence, this study showed that secondarily treated domestic wastewater could potentially be a suitable and sustainable medium for microalgae grown to be used as biodiesel feedstock.